MDCK Cell :Reply

In article <35ED6316.7DE0 at med.unc.edu>, stigh at med.unc.edu says...
>>°­ ¹Ì ¼± wrote:
>>>> I culture MDCK cell to research toxicity mechaism of heavy metal.
>> MDCK cell is kidney epithelial cell and always had grown very quickly in
my
>> lab. Because it bind to each other by tight junction, gap junction,
>> desmosome and focal adhesion, it looks like very big aggregates. But
Shape
>> of mdck cell changed into fibroblastic and size of the cell looks very
>> small. All the worse, after trypsinizing to subculture, only half the cell
>> survive. Doubling time of the cell is very long.
>> I want to knowf what happen to my cell!!
>> Could character of MDCK cell be changed by continuous subculture?
>>>> please let me know.
>> thank you in advance.
>>>> email to oasis at cau.ac.kr>>>> Mi Sun Kang
>> Department of Environmental and Health Chemisty,
>> College of Pharmacy,Chung-Ang university,seoul, south korea
>>Dr. Kang,
> I have not had that much experience with the MDCK cell line but
>have worked with a number of other cell lines and primary cultures over
>the years. I'm not aware of the MDCK spontaneously transforming in
>vitro, fom epithelial to fibroblast. Have you had your cell cultures
>checked for Mycoplasma/Ureaplasma contamination? The condition of your
>cells makes me think that the cell line is contaminated either through
>subpassaging or a component of the medium, i.e. serum. Bear in mind that
>these bacteria will not be visible by routine phase contrast light
>microscopy or histological/Gram stain. If your cells are not
>contaminated with bacteria or virus then there may be a cytotoxic
>component in your culture conditions, i.e. medium, plastics, water,
>glassware etc. which will be difficult to identify or never will be. An
>additional but less likely possibility is that you MDCK cell line was
>cross contaminated with another cell type being used in your laboratory.
>> In my lab. cell cultures are routinely checked for
>contamination every 6 weeks (Hoechst stain method) and discarded when
>they reach 50-60 subpassages from the freezer. This strategy ensures
>that there are always clean, viable cell stocks in the freezer, and that
>if there are problems they can be isolated and identified. Where
>possible we have specific markers (cytokeratins, receptors) for specific
>cells to ensure identity and continuity.
> I hope that this advice is useful and that your problem can be
>resolved.
>Stephen Knight
>Microbiology and Immunology
>UNC- Chapel Hill, North Carolina
Excellent answer and any of these process could very well be the cause of his
problems.
Another distinct possibility is that he has tried to cultivate the cell line
beyond it's normal Hayflick limit. The loss of cells, length between
doublings and inappropriate phenotype expression all seem to point to cells
in their senescent stage of development.
A test for telomeric length on the chromosomes could either confirm or
eliminate this possibility.
Thomas Mahoney, Pres.
Lifeline Laboratories, Inc.
http://home.earthlink.net/~excelife/index.html